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Aims:Chitosan and epidermal growth factor (EGF) have been shown to improve wound healing. This study investigates the healing effects of a spray solution (NewEpi, JoyCom Bio-Chem Co. Ltd., Taiwan) containing recombinant human EGF (rhEGF) delivered via a newly patented technology—chitosan microencapsulated nanoparticles.Methods:On Wistar rats, two full-thickness wounds on the dorsum bilateral of the spine were created. The rats were randomised to the following treatment groups: hydrogel, wet dressing, foam, rhEGF spray and rhEGF spray+foam. Sterile dressings were applied and changed daily. A total of 2μg of rhEGF was administered in two sprays during each dressing change. All animals were euthanised on day 14. Tissue samples were taken from the wound bed, including an area of 2cm surrounding the wound margin for histological evaluations.Results:Wounds treated with the rhEGF spray achieved the greatest size reduction by day 14 compared with other types of conventional dressings. An overall significant difference in levels of collagen synthesis existed between groups (p<0.01). Pair-wise comparisons showed that the rhEGF spray treatment significantly promoted higher levels of mature Type I collagen than any other conventional dressings (p<0.01), whereas non-rhEGF treatments resulted in higher levels of Type III collagen. The regenerated tissue in rhEGF spray treatment groups was also in alignment with that of normal skin. Epidermis, dermis and hair follicles were easily observed in wounds treated with the rhEGF spray.Conclusion:The major challenge of topical application of rhEGF was overcome by using a new drug delivery technology: chitosan–rhEGF nanoparticles. The positive healing effects observed in this study suggest the therapeutic potentials of this novel rhEGF topical spray solution.

Pulmonary fibrosis is a progressive diffuse parenchymal lung disorder with a high mortality rate. Studies have indicated that injured lung tissues release various pro-inflammatory factors, and produce a large amount of nitric oxide. There is also accumulation of collagen and oxidative stress-induced injury, collectively leading to pulmonary fibrosis. Antrodia cinnamomeais an endemic fungal growth in Taiwan, and its fermented extracts exert anti-inflammatory effects to alleviate liver damages. Hence, we hypothesized and tested the feasibility of using A. cinnamomeaextracts for treatment of pulmonary fibrosis.

Smoker patients with non-small cell lung cancer (NSCLC) have poorer prognosis and survival than those without smoking history. However, the mechanisms underlying the low response rate of those patients to EGFR tyrosine kinase inhibitors (TKIs) are not well understood. Here we report that exposure to cigarette smoke extract enhances glycolysis and attenuates AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR; this in turn reduces the sensitivity of NSCLC cells with wild-type EGFR (EGFRWT) to EGFR TKI by repressing expression of liver kinase B1 (LKB1), a master kinase of the AMPK subfamily, via CpG island methylation. In addition, LKB1 expression is correlated positively with sensitivity to TKI in patients with NSCLC. Moreover, combined treatment of EGFR TKI with AMPK activators synergistically increases EGFR TKI sensitivity. Collectively, the current study suggests that LKB1 may serve as a marker to predict EGFR TKI sensitivity in smokers with NSCLC carrying EGFRWTand that the combination of EGFR TKI and AMPK activator may be a potentially effective therapeutic strategy against NSCLC with EGFRWT.

Objective:To compare the healing results between platelet-rich plasma (PRP) and platelet-derived patches versus traditional advanced wound dressings in patients with chronic wounds.Method:Patients with and without diabetes were divided into two groups, each of which received either PRP patch treatments or the advanced wound dressings. All wounds were cleaned, debrided and assessed by physicians. The data were analysed and represented as mean ± standard deviation (SD). Student's t-test was used to calculate the significance of differences between both groups. Values of p<0.05 were considered statistically significant.Results:Patients with and without diabetes receiving PRP patch treatments saw improvement in wound healing in two weeks (p=0.0083). Patients with diabetes who received platelet-derived patch treatment and PRP injection experienced wound size reduction to <25% of the original area by the fourth week of treatment, and >90% of the subjects had wounds of <10% their original size in the last three weeks of the trial. Conversely, the wound area in the control subjects receiving traditional advanced wound dressings remained at 25–50% of their original size from the fourth week of treatment to the end of the trial. The healing process of the PRP patch experimental group was statistically significant compared with the control group (p<0.0001).Conclusion:Combining treatments of PRP injections and platelet-derived patches significantly improved the healing outcomes of patients with chronic wounds, most notably in patients with diabetes, when compared with a traditional treatment of advanced wound dressings.

Extracellular superoxide dismutase 3 (SOD3), one member of the antioxidant defense system and a superoxide scavenger, has been noted to be downregulated in the kidneys of diabetic mice and is characterized by a heparin-binding domain that can anchor the protein to the endothelium and extracellular matrix. The association of the serum and urinary SOD3 levels with diabetic nephropathy in different stages has never been evaluated. It remains unclear how urinary SOD3 changes in different renal diseases. We recruited 98 Taiwanese patients with type 2 diabetes and 10 patients with early chronic kidney disease (CKD) into this study. Biochemical analyses were performed, including evaluation of the serum SOD3, urinary SOD3, urinary albumin, urinary vascular endothelial growth factor (VEGF), and urinary angiotensinogen (ANG). The Kruskal-Wallis rank sum test was used to compare various parameters among the three groups of patients: early CKD, diabetes alone, and diabetes with CKD. Results showed that lower serum and urinary SOD3 levels were observed in the group of patients with diabetes alone. Higher serum and urinary SOD3 levels were observed in the group of patients with diabetes and CKD, which had higher albuminuria and serum creatinine levels. The serum SOD3 levels were significantly positively correlated with renal function, according to the serum creatinine level. The urinary levels of SOD3 were significantly correlated with other urinary biomarkers such as urinary ANG and VEGF. Furthermore, albuminuria can positively predict the serum SOD3 level for the ratio of urinary albumin to urinary creatinine (ACR) >1,190.769 mg/g and the urinary SOD3 level for ACR ≥300 mg/g.

Mesenchymal stem cells (MSCs) are widely considered for treatment of pulmonary fibrosis based on the anti‐inflammatory, antifibrotic, antiapoptotic, and regenerative properties of the cells. Recently, elevated levels of oncostatin M (OSM) have been reported in the bronchoalveolar lavage fluid of a pulmonary fibrosis animal model and in patients. In this work, we aimed to prolong engrafted MSC survival and to enhance the effectiveness of pulmonary fibrosis transplantation therapy by using OSM‐preconditioned MSCs. OSM‐preconditioned MSCs were shown to overexpress type 2 OSM receptor (gp130/OSMRβ) and exhibited high susceptibility to OSM, resulting in upregulation of the paracrine factor, hepatocyte growth factor (HGF). Moreover, OSM‐preconditioned MSCs enhanced cell proliferation and migration, attenuated transforming growth factor‐β1‐ or OSM‐induced extracellular matrix production in MRC‐5 fibroblasts through paracrine effects. In bleomycin‐induced lung fibrotic mice, transplantation of OSM‐preconditioned MSCs significantly improved pulmonary respiratory functions and downregulated expression of inflammatory factors and fibrotic factors in the lung tissues. Histopathologic examination indicated remarkable amelioration of the lung fibrosis. LacZ‐tagged MSCs were detected in the lung tissues of the OSM‐preconditioned MSC‐treated mice 18 days after post‐transplantation. Taken together, our data further demonstrated that HGF upregulation played an important role in mediating the therapeutic effects of transplanted OSM‐preconditioned MSCs in alleviating lung fibrosis in the mice. StemCellsTranslationalMedicine2017;6:1006–1017

Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.

Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs) have been efficiently achieved by somatic cell nuclear transfer (SCNT). We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/−) mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/−cells exhibit naive pluripotency as evidenced by generation of Terc+/−ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.

Background Although epidemiological and laboratory studies report that chronic inflammatory conditions contribute to the pathogenesis of cancer, it remains controversial whether chronic rhinosinusitis (CRS) results in nasopharyngeal cancer (NPC).Methods Retrospective cohort study was performed from the National Health Insurance (NHI) Taiwan database. This study prospectively examined whether CRS or nasal polyposis is associated with NPC risk in the NHI, a population-based cohort of 231,490 Taiwan Chinese individuals with a mean age of 32 years, recruited between 2000 and 2006. We collected information from the Longitudinal Health Insurance Database. Each subject completed an interview including questions about medical conditions, and the NPC occurrence and survival statuses were determined by linkage to population-based NHI registries in Taiwan. In addition, each NPC and CRS subject had completed an interview on medical condition to confirm their diagnosis.Results After adjustment for age, sex, hypertension, diabetes mellitus, allergic rhinitis, otitis media, coronary artery disease, pharyngitis, and tonsillitis, individuals with rhinosinusitis were found to have a 3.55-fold increased risk of developing NPC compared with individuals without rhinosinusitis (hazard ratio = 3.55; 95% CI = 2.22–5.69). The same results were also observed when the study subjects were analyzed without comorbidities.Conclusion Adult patients with rhinosinusitis should be followed up with regard to the nasopharynx for at least 3 years, particularly repeat sinusitis patients.

Open tibial shaft fractures are the most common open fractures, and many complications can occur. During the treatment period, infection leading to osteomyelitis was the most common complication. However, no consensus exists regarding the ideal management for such cases in the literature.The purposes of this retrospective study were to review the treatment of patients with chronic tibial shaft osteomyelitis over the past 14 years who were referred to the authors’ institution and to provide a staged protocol for spontaneous wound healing. The staged protocol included: (1) radical debridement for infected bone and soft tissue; (2) immediate application of Ilizarov’s apparatus for all patients except those needing delayed application; (3) osteotomy in healthy bone; (4) simultaneous distraction–compression osteogenesis and histogenesis; (5) additional docking-site bone grafting; and (6) shifting the external fixator to a locked nail when callus formation was visible at the distraction site. Union was achieved in 15 of 16 patients, with an average external fixation time of 4.5 months (range, 3–6 months). No deformity or leg-length discrepancy greater than 1 cm occurred.In the treatment of chronic osteomyelitis, this staged protocol was safe and successful and allowed for union, realignment, reorientation, and leg-length restoration. Regarding the soft tissues, this technique provides a unique type of reconstructive closure for infected wounds. It is suggested that the staged protocol is reliable in providing successful simultaneous reconstruction for bone and soft tissue defects without flap coverage.

Managing refractory osteomyelitis around the ankle joint has been challenging. Destruction of both the ankle and the subtalar joints was common in cases of open fracture. For those who already had multiple surgeries, it would be tough to salvage the limb. Our goal was to set up a staged surgical protocol aiming in treating the aforementioned clinical issue.

We evaluated the effect of revision with dynamic compression plate (DCP) and cancellous bone graft for aseptic nonunion after surgical treatments of humeral shaft fracture.

OBJECTIVE To evaluate the role of leptin, a 16-kDa adipocyte-derived hormone, in the development of metabolic dysregulation of psoriasis. DESIGN Case-control study. SETTING Referral centers. PATIENTS Seventy-seven patients with psoriasis and 81 age- and sex-matched control subjects were included in the study. INTERVENTION Enzyme-linked immunoassay of serum samples from study subjects. MAIN OUTCOME MEASURES Serum leptin levels and proportions of comorbidities (including hypertension, diabetes mellitus, metabolic syndrome, hypertriglyceridemia, and reduced high-density lipoprotein cholesterol concentrations) in cases vs controls were compared using χ2 and Mann-Whitney tests. The clinical significance of leptin in psoriasis was analyzed using logistic regression models. RESULTS Significantly more obesity (odds ratio [OR], 2.67) and hypertension (2.17) (P =.04 for both) were observed in subjects with psoriasis. High serum leptin levels (≥7397.43 pg/mL) were found in female subjects (OR, 6.05; P &lt; .001) and in subjects with obesity (3.45; P =.01), hypertension (2.19; P =.04), metabolic syndrome (3.58; P =.008), and psoriasis (2.25; P =.02). On multivariate analysis, psoriasis (OR, 4.57; P =.009) was significantly associated with hyperleptinemia independent of female sex (26.36; P &lt; .001), metabolic syndrome (4.37; P =.04), and obesity (2.83; P =.12). Finally, patients with psoriasis who had hyperleptinemia tended to be female (P &lt; .001) and manifested obesity (P =.002) and metabolic syndrome (P =.003). CONCLUSIONS Hyperleptinemia is associated with psoriasis independent of female sex and other conventional cardiovascular risk factors such as obesity and metabolic syndrome. Hyperleptinemia in psoriasis may contribute to metabolic syndrome.Arch Dermatol. 2008;144(12):1571-1575--

Krüppel-like factor 4 (KLF4) is a transcription factor regulating cell growth and differentiation. It has been postulated as a tumor suppressor gene in several tumors including colon cancer and prostate cancer; whereas, it is activated in other tumors such as breast cancer and oropharyngeal carcinomas. To understand the significance KLF4 on clinicopathologic implications in human squamous cell carcinoma skin (SCC) progression, thirty six formalin-fixed paraffin-embedded samples of cutaneous SCC and 28 samples of Bowen's disease were stained with anti-KLF4 antibodies. A semiquantitative evaluation of the degree of staining intensity and localization of expression were analyzed and correlated with histological and clinical features. Nuclear KLF4 staining was seen in differentiated epithelium including suprabasal keratinocyte of non-lesional skin. Strong nuclear KLF4 staining was observed in tumor parts of both SCC and Bowen's disease, when compared with their non-tumor parts (P=0.001). Increased expression of KLF4 protein and mRNA were found in squamous cell carcinoma cell line studies and fresh skin tissue respectively, using western blotting and semi-quantitative real time polymerase chain reaction (PCR). SCC with moderately and poorly differentiated tumors tend to have maturation independent (MI) staining pattern (p=0.0004), compared to maturation dependent (MD) staining in well-differentiated tumors. Finally, constitutive nuclear KLF4 staining pattern was significantly associated with moderately and poorly differentiated tumors (p=0.003), and tumor metastasis (p=0.024). We propose that KLF4 expression is associated with human skin SCC progression and metastases.

We evaluate the effect of reconstructing huge defects (mean, 15.8 cm) of the distal femur with Ilizarov's distraction osteogenesis and free twin-barreled vascularized fibular bone graft (TVFG).

A new internal fixation concept for the supracondylar femoral fracture after total knee arthroplasty (TKA) was investigated. In the new design, the intramedullary nail was fixed on the modified femoral component of the total knee prosthesis by a locking screw. The new internal fixator complex should have greater structural stiffness by increasing the structure contact area between bone and implant. To test this improvement, biomechanical tests were conducted to compare the mechanical properties (axial and torsional stiffness) of the fractured femurs that were stabilized by three different fixators (DCS plate, GSH nail and new design fixator). The compressive test and torsional test were done by the MTS Bionix858 to compare the mechanical stability of these fixators. The results showed no significant difference among groups in compressive stiffness, which meant that these three fixators could stabilize the fracture effectively in the longitudinal direction. The torsional stiffness of all three fixation groups were lower than the Intact group with significant difference. But the torsional stiffness of the new designed fixator was higher than the DCS plate and the GSH nail. The results supported the new design concept's practicality. The new design also has better initial stability in the fixation of a supracondylar femoral fracture after TKA.

Hypermethylation of multiple CpG islands is a common event in cancer. To assess the prognostic values of this epigenetic alteration, we developed Methylation Target Array (MTA), derived from the concept of tissue microarray, for simultaneous analysis of DNA hypermethylation in hundreds of tissue genomes. In MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers. A panel of 468 MTA amplicons, which represented the whole repertoire of methylated CpG islands in 93 breast tumors, 20 normal breast tissues, and 4 breast cancer cell lines, were arrayed on nylon membrane for probe hybridization. Positive hybridization signals detected in tumor amplicons, but not in normal amplicons, were indicative of aberrant hypermethylation in tumor samples. This is attributed to aberrant sites that were protected from methylation-sensitive restriction and were amplified by PCR in tumor samples, while the same sites were restricted and could not be amplified in normal samples. Hypermethylation frequencies of the 10 genes tested in breast tumors and cancer cell lines were 60% for GPC3, 58% for RASSF1A, 32% for 3OST3B, 30% for HOXA5, 28% for uPA, 25% for WT1, 23% for BRCA1, 9% for DAPK1, and 0% for KL. Furthermore, hypermethylation of 5 to 7 loci of these genes was significantly correlated with hormone receptor status, clinical stages, and ages at diagnosis of the patients analyzed. This novel approach thus provides an additional avenue for assessing clinicopathological consequences of DNA hypermethylation in breast cancer.

Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine α-lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The αLA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 μg/ml, over 35–200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.

Swine are reservoirs of hepatitis E virus (HEV). In this study, a 2‐year survey of HEV in feces and sera of swine was conducted to determine if: 1) HEV has circulated among pigs for some time in Taiwan; 2) the spread of HEV among different‐aged pigs; and 3) there exists HEV strains possibly imported through trading. From 1998–2000, 521 serum samples and 54 fecal specimens from pigs were examined by reverse transcription polymerase chain reaction. None of the 11 pigs in suckling stage (< 2 months) were serum HEV RNA positive. The highest viremia rate (4.5%) was in pigs of 2 months age, followed by 1.2% and 1.8% in pigs of growing (3–4 months) and finishing stages (5–6 months), and none in pigs older than 6 months. Viremia showed little variation in different years and areas. None of the 20 fecal samples from pigs in suckling stage were HEV RNA positive, whereas 9% of the 34 samples from pigs in growing or finishing stages were positive. Most swine HEV isolates in Taiwan clustered within the genotype 4, whereas the three HEV isolates cloned from pigs imported recently from the U.S. belonged to the genotype 3 HEV in the U.S. The results suggest that HEV may infect pigs at an early growing stage and spread unnoticed among pigs and possibly across countries through trading. J. Med. Virol. 66:488–492, 2002. © 2002 Wiley‐Liss, Inc.

Oligonucleotide microarray-based hybridization is an emerging technology for genome-wide detection of DNA variations. We have extended this principle and developed a novel approach, called methylation-specific oligonucleotide (MSO) microarray, for detecting changes of DNA methylation in cancer. The method uses bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. The amplified product, therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. A test sample is hybridized to a set of oligonucleotide (19-23 nucleotides in length) arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions, and quantitative differences in hybridization are determined by fluorescence analysis. A unique control system is also implemented to test the accuracy and reproducibility of oligonucleotides designed for microarray hybridization. This MSO microarray was applied to map methylated CpG sites within the human estrogen receptor alpha (ERalpha) gene CpG island in breast cancer cell lines, normal fibroblasts, breast tumors, and normal controls. Methylation patterns of the breast cancer cell lines, determined by MSO microarray, were further validated by bisulfite nucleotide sequencing (P <0.001). This proof-of-principle study shows that MSO microarray is a promising technique for mapping methylation changes in multiple CpG island loci and for generating epigenetic profiles in cancer.

In nonendemic areas, most patients with acute hepatitis E were infected through traveling to endemic areas. However, some patients did not have a history of foreign travel before infection. Furthermore, high seroprevalence rates of antibody to hepatitis E virus (anti‐HEV) were found in the general adult population in some countries without any recorded outbreak of hepatitis E. The significance of anti‐HEV assay in these subjects remains obscure. To study if swine might be a source of HEV infection, HEV was tested in sera of 235 pigs in Taiwan, and from 5 patients with acute HEV infection who either denied or did not provide any foreign travel history. Three (1.3%) pigs had detectable swine HEV RNA. The swine and human HEV strains from Taiwan formed a monophyletic group, distinct from three previously reported groups: the United States human and swine HEV strains, the Mexico strain, and the largest group composed of the Asian and the African strains. The identity of nucleotide sequences was 84–95% between swine and human HEV strains in Taiwan, and 72–79% between Taiwan strains and those from different areas. The predicted amino acid sequence of a Taiwan swine HEV strain within the peptide 3‐2 used in commercial anti‐HEV assay showed a high identity (91–94%) with those of other human and swine HEV strains. Swine may be a reservoir of HEV and subclinical swine HEV infection may occur. Cross‐reactivity of current anti‐HEV assay may account for the high prevalence rate of anti‐HEV in the general population in nonendemic areas. J. Med. Virol. 60:166–171, 2000. © 2000 Wiley‐Liss, Inc.

ABSTRACTThe aim of the present study was to investigate the expression activity, both in vitroand in vivo, of the porcine growth hormone complementary DNA (pGH cDNA) in porcine fetal fibroblast (PFF) cells. The pGH gene had been constructed inside the bicistronic retroviral vector PSN and subsequently transfected into PFF cells further encapsulated with immunoprotective microcapsules. This would provide a way to evaluate the improvement in growth performance of Tao-Yuan swine by the use of nonautologous microencapsulated fibroblasts carrying the pGH cDNA via the technique of somatic gene therapy. Results from Southern blot analysis confirmed that the full length of the pGH cDNA was completely integrated into the genome of the PFF cells after they had been infected one to four times using a PSN retroviral vector. Moreover, Northern blot analysis showed that high transcription activity was present in clones infected twice, and exogenous pGH secretion was found when the pGH-infected PFF had been further cultured for 48 hr in vitroand subjected to immunoblot assay. Encapsulation of the pGHPFF with an alginatepoly-l-lysinealginate membrane did not show any deterioration in their proliferation and survival both in vitroand in vivo. The pGH gene in encapsulated recombinant fibroblasts was fully expressed after it had been transplanted into the peritoneal cavity of the Tao-Yuan swine, and reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed on the microcapsules retrieved 1 month later. The feasibility of pGH gene therapy to improve midget Tao-Yuan swine growth enhancement is further supported by the fact that transplantation of the encapsulated recombinant fibroblast cells resulted in a much more significant increase in weight gain than in those swine in either the age-matched untreated control group or in those that had been transplanted with uncapsulated recombinant PFF cells (10.56 1.01 kg versus 6.95 0.94 and 5.27 1.30 kg; p< 0.05). These experimental data suggest that growth hormone gene therapy did provide an alternative approach for growth improvement in midget Tao-Yuan swine.

Exogenous DNA microinjected into one-cell mouse zygotes either integrates into the host genome within a short time span, or is rapidly degraded. On integration, a transgene squence is frequently reiterated. In this report, we describe the enzymatic amplification analysis of transgene junctions of 12 transgenic mice carrying different copy numbers of the same transgene with dissimilar ends. The transgene was composed of the regulatory sequence of the type 18 human papillomavirus linked to the TAg gene of the SV40 virus. Nucleotide sequences of 36 of these junctions were also determined. Deletions were found in 33 (91.7%) of the junctions analysed. At the crossover regions, 55.6% contained short overlapping sequences of one to six nucleotides. Insertions of 2–6 extraneous nucleotides were also found in 8.3% of the transgene junctions. Within a 10-nucleotide sequence on both sides of the transgene junctions, topoisomerase I (topo I) cleavage sites, runs of homogeneous purines or pyrimidines, alternating purine-pyrimidine tracks and (A-T)-rich sequences were found frequently. Stringent control experiments were also performed to ascertain that the observations made were not artefacts resulting from the polymerase chain reaction. Our data therefore indicate that damage had occurred quite frequently and extensively in our transgene construct. Such transgene damage may also occur to various extents in mice carrying other transgenes. Primary structure of the nucleotide sequences of the injected DNA seems to influence the process of transgene reiteration and aberration.

We elucidate the effect of surgical treatment of acute basicervical fractures of femur by closed reduction and internal fixation with dynamic hip screw (DHS).

Steroidogenic acute regulatory protein (StAR protein) has been believed playing important role in steroid hormone production. Our previous results indicate that protein expression and kinase activity of cyclin-dependent kinase 5 (Cdk5) are under luteinizing hormone receptor/cAMP control and regulates mouse androgen production through StAR protein phosphorylation and stabilization. The aim of this study is to understand that whether the Cdk5 regulation on androgen production can be happened in ovary thecal cells and responds to their androgen production. This is the pioneer report indicating the physiological function of Cdk5 in ovary cells. The results showed that Cdk5 and its regulator, p35, were abundant in primary mouse thecal cells. Furthermore, we found that the protein expressions of Cdk5 and p35 and kinase activity of Cdk5 in thecal cells were all controlled by human chorionic gonadotropin (hCG). Taking advantage of cAMP-related blockers, we identified that cAMP-related pathway was involved in hCG-dependent regulation on Cdk5/p35 in thecal cells. Specific Cdk5 inhibitor could significantly decrease the phosphorylation and protein levels of StAR in thecal cells. Finally, Cdk5 inhibitor could also diminish androstenedione production in thecal cell culture medium. In conclusion, these results first elucidate the physiological function of Cdk5 in steroid production of mouse ovary.(poster)

To study the prevalence and type of the hepatitis B e antigen (HBeAg)-negative mutant in hepatitis D virus (HDV) superinfection, the precore region of hepatitis B virus (HBV) was analyzed by cycle sequencing. Of the 58 samples sequenced, 24 were wild type and 34 carried mutants. The precore stop mutation (TAG) at the 28th codon was found in 32 cases, other mutations were found in 7, and double mutations were found in 5. The absence of HBeAg showed a substantial agreement with the presence of mutants (κ value, 0.74). Of the acute hepatitis patients, HDV replication and clinical manifestations were not significantly different between those with mutant and wild type virus, except that those with mutant virus were older (mean age, 48 vs. 28 years; P < .002). The absence of HBeAg in these patients is mainly due to HDV superinfection in older HBV carriers who already had precore mutant.